Instant Volume Microscopy of Organoids with SOLIS

Advancements in microscopy techniques have revolutionized our ability to explore the intricacies of biological systems, with engineered human heart tissue (EHT) being a particularly challenging target. In this article, we will have an in-depth look at the award-winning SOLIS technique (scanned oblique light-sheet instant-volume sectioning), a new twist on multifocus fluorescence microscopy. Through its unique capabilities, SOLIS offers a new approach on organoid and tissue imaging, providing unprecedented insights into the cellular architecture of these complex artificial samples. By being able to record optically sectioned volumes during single camera exposures, SOLIS demonstrates remarkable advantages over traditional multifocus microscopy, underscoring its potential to transform our understanding of developmental biology, disease mechanisms, and potential therapeutic interventions

A joint Richardson-Lucy deconvolution algorithm for the reconstruction of multifocal structured illumination microscopy data.

We demonstrate the reconstruction of images obtained by multifocal structured illumination microscopy, MSIM, using a joint Richardson-Lucy, jRL-MSIM, deconvolution algorithm, which is based on an underlying widefield image-formation model. The method is efficient in the suppression of out-of-focus light and greatly improves image contrast and resolution. Furthermore, it is particularly well suited for the processing of noise corrupted data. The principle is verified on simulated as well as experimental data and a comparison of the jRL-MSIM approach with the standard reconstruction procedure, which is based on image scanning microscopy, ISM, is made. Our algorithm is efficient and freely available in a user friendly software package.This work was supported by grants from the Leverhulme Trust, the Engineering and Physical Sciences Research Council, UK (grant EP/H018301/1) and by the Medical Research Council (grant MR/K015850/1). FS wishes to acknowledge support from the Studienstiftung des deutschen Volkes and the Erlangen Graduate School in Advanced Optical Technologies (SAOT) by the German Research Foundation (DFG).This was originally published in Methods and Applications in Fluorescence (F Ströhl, CF Kaminski, Methods and Applications in Fluorescence 2015, 3, 014002

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors.

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.This work was supported by grants from the Leverhulme Trust, the Engineering and Physical Sciences Research Council [EP/H018301/1, EP/G037221/1]; Alzheimer Research UK [ARUK-EG2012A-1]; Wellcome Trust [089703/Z/09/Z] and Medical Research Council [MR/K015850/1, MR/K02292X/1]. We thank K. O’Holleran for assistance with the design of the microscope, and L. Shao and R. Heintzmann for useful discussions and suggestions

Arbeitsbericht Nr. 2020-03, September 2020

Fehlende Werte stellen in zahlreichen praktischen Anwendungen vie-mehr den Regelfall als eine Ausnahme dar, erweisen sich aber bei vielen statistischen Verfahren als störend. Die vorliegende Studie untersucht die Auswirkungen von fehlenden Werten auf die Ergebnisse der multiplen linearen Regression. Dazu werden zunächst spezielle Formen von fehlenden Daten und ausgewählte Verfahren zum Umgang mit diesen vorgestellt. Im Rahmen einer Simulationsstudie werden anschließend die Auswirkungen von verschiedenen Ausfallquoten und -mechanismen anhand von sechs empirischen Datensätzen untersucht. Neben einer Analyse verschiedener Einflussgrößen erfolgt ein Vergleich der vorgestellten Verfahren zur Behandlung der fehlenden Werte. Es zeigt sich, dass keines der untersuchten Verfahren allen anderen Verfahren in jeder Hinsicht überlegen ist und die Wahl des „besten“ Verfahrens von der Struktur des Datensatzes und der späteren Verwendung der Regressionsfunktion abhängt. Darüber hinaus konnte festgestellt werden, dass eine Erhöhung der Ausfallquote im Allgemeinen zu einer Verschlechterung der Ergebnisse führt. Die Einflüsse der Objekt- und Merkmalsanzahl hängen von dem jeweiligen Verfahren und den weiteren Eigenschaften des Datensatzes ab und sollten stets zusammen betrachtet werden

Integration von Gewichtsrestriktionen in das DEA-Modell nach Charnes, Cooper und Rhodes: exemplarische Optionen und Auswirkungen

Ziel des Beitrags ist es, die Integration von Gewichtsrestriktionen in das DEA-Basismodell nach Charnes, Cooper und Rhodes (CCR-Modell) zu untersuchen. Für ein fiktives Beispiel werden die Ergebnisse der um verschiedene Gewichtsrestriktionen erweiterten DEA-Modelle präsentiert und diskutiert. In einfachen Sensitivitätsanalysen wird der Einfluss ausgewählter Gewichtsrestriktionen auf die Effizienzwerte näher beleuchtet

Flat-Field Super-Resolution Localization Microscopy with a Low-Cost Refractive Beam-Shaping Element.

Super-resolution single-molecule localization microscopy, often referred to as PALM/STORM, works by ensuring that fewer than one fluorophore in a diffraction-limited volume is emitting at any one time, allowing the observer to infer that the emitter is located at the center of the point-spread function. This requires careful control over the incident light intensity in order to control the rate at which fluorophores are switched on; if too many fluorophores are activated, their point-spread functions overlap, which impedes efficient localization. If too few are activated, the imaging time is impractically long. There is therefore considerable recent interest in constructing so-called 'top-hat' illumination profiles that provide a uniform illumination over the whole field of view. We present the use of a single commercially-available low-cost refractive beamshaping element that can be retrofitted to almost any existing microscope; the illumination profile created by this element demonstrates a marked improvement in the power efficiency of dSTORM microscopy, as well as a significant reduction in the propensity for reconstruction artifacts, compared to conventional Gaussian illumination

A Protocol for Single-Molecule Translation Imaging in Xenopus Retinal Ganglion Cells

Single-molecule translation imaging (SMTI) is a straightforward technique for the direct quantification of local protein synthesis. The protein of interest is fused to a fast-folding and fast-bleaching fluorescent protein, allowing one to monitor the appearance of individual fluorescence events after photobleaching of pre-existing proteins in the cell under investigation. The translation of individual molecules is then indicated by photon bursts of sub-second length that appear over a dark background. The method thus shares attributes with fluorescence recovery after photobleaching (FRAP) microscopy. Resulting datasets are similar to those generated by localization-based super-resolution microscopy techniques and can be used both to generate density maps of local protein production and to quantify the kinetics of local synthesis. The detailed protocol described in this chapter uses a Venus-β-actin fusion construct to visualize and measure the β-actin mRNA translational activity in Xenopus retinal ganglion cell growth cones upon Netrin-1 stimulation, which can be readily adapted for detecting translation events of other mRNAs in various cell types

Label-free nanoscopy enabled by coherent imaging with photonic waveguides

SPIE Article-Sharing Policies https://www.spiedigitallibrary.org/article-sharing-policiesIn this project it was found that Fourier ptychographic microscopy can be improved far beyond its conventional limits via waveguide-based optical systems. Extensive in silico studies showed that images obtained on highrefractive index material waveguide chips in conjunction with hyperspectral illumination light and finely designed waveguide geometries can be combined via a modified phase-retrieval algorithm to yield a resolution below 150 nm

Super-condenser enables labelfree nanoscopy.

Labelfree nanoscopy encompasses optical imaging with resolution in the 100 nm range using visible wavelengths. Here, we present a labelfree nanoscopy method that combines coherent imaging techniques with waveguide microscopy to realize a super-condenser featuring maximally inclined coherent darkfield illumination with artificially stretched wave vectors due to large refractive indices of the employed Si3N4 waveguide material. We produce the required coherent plane wave illumination for Fourier ptychography over imaging areas 400 μm2 in size via adiabatically tapered single-mode waveguides and tackle the overlap constraints of the Fourier ptychography phase retrieval algorithm two-fold: firstly, the directionality of the illumination wave vector is changed sequentially via a multiplexed input structure of the waveguide chip layout and secondly, the wave vector modulus is shortend via step-wise increases of the illumination light wavelength over the visible spectrum. We test the method in simulations and in experiments and provide details on the underlying image formation theory as well as the reconstruction algorithm. While the generated Fourier ptychography reconstructions are found to be prone to image artefacts, an alternative coherent imaging method, rotating coherent scattering microscopy (ROCS), is found to be more robust against artefacts but with less achievable resolution